A porcine in vitro assay for renal cortical 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) and 25-hydroxyvitamin D3- 24-hydroxylase (24-hydroxylase) activities has been developed. This will be the first opportunity for investigators to study the regulation of these enzymes in vitro in a mammalian species. Previous in vitro studies have used birds, which have a unique physiology that may not be representative of mammals. In vivo studies in rats and plasma 1,25-(OH)2D3 levels have given conflicting results. The pig was chosen because of its physiologic similarity to humans. We have preliminary data that 1-hydroxylase and 24-hydroxylase activities can be demonstrated in porcine renal cortical homogenates and slices. The latter will allow polypeptide and protein hormones thought to play a role in regulation 1,25-(OH)2D3 production to be tested. Growth hormone, insulin, prolactin and parathyroid hormones will be investigated because of confusion in the literature regarding their actions. Somatomedin will be tested because it may mediate growth hormone's actions and because it has insulin-like activity. Insulin's action is important because low 1,25-(OH)2D3 plasma levels may be related to the osteopenia of diabetes mellitus. Growth hormone and somatomedin will be studied because they may be related to higher 1,25-(OH)2D3 plasma levels associated with human growth and animal growth intestinal calcium adsorption during pregnancy and lactation in mammals. The hydroxylase activities will be measured by the in vitro conversion of (3H)-25-OH-D3 to (3H)-1,25-(OH)2D3 and (3H)-24,25-(OH)2D3. The dihydroxy metabolites will be isolated by Sephadex LH-20 chromatography followed by high pressure liquid chromatography. In addition to studying the direct effects of hormones on cortical slices, the effect of in vitro hormone manipulations on in vitro hydroxylase activities will be investigated and compared to measurements of plasma Ca, P, 1,25-(OH)2D3, 24,25-(OH)2D3 and pH. The in vivo production rates of both metabolites will be compared with the in vitro rates to relate in vitro to in vivo.